We will develop a novel approach to the investigation of metabolites in genetically defined biological systems, which we term "isotope-assisted differential metabolomics". We will use the model plant Arabidopsis, which can be grown economically on inexpensive labeled precursors, as the source for tissue containing complete metabolomes with four (4) different isotopic compositions: natural abundance, carbon-13, nitrogen-15, and carbon-13 plus nitrogen-15. Labeling with stable isotopes will be close to 100% so that single isotopomers need only be considered. These four (4) tissue sources will be used as reagents in experiments designed (1) to identify the major metabolites present in the tissue, (2) to determine structures of novel metabolites by nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS), and (3) to identify the minimal conditions of extraction and fractionation needed to enable concentrations of individual metabolites to be determined from MS analysis in which labeled metabolites are used as intensity standards. The labeled metabolomes will serve as the source for heavy metabolites used in quantifying levels of molecules in unknown samples; in addition, they will assist in the analysis of mass peak overlaps. The double-labeled metabolites will enable application to metabolomics of the multinuclear, multidimensional NMR technology that has proved so valuable in the fields of protein and nucleic acid NMR. Supporting technologies to be developed include specialized laboratory information management and database software (created in house from existing software tools), MS instrumentation designed to overcome sensitivity and suppression problems associated with mass spectrometry of complex mixtures of metabolites, and instrumentation and data collection and analysis methodology for NMR. This strategy and associated new technology will be tested and refined through differential metabolomics investigations of (1) Arabidopsis thaliana and its single-gene knockouts and (2) genetically defined mouse models for type-2 diabetes.